Abstract
p97 is a "segregase" that plays a key role in numerous ubiquitin (Ub)-dependent pathways such as ER-associated degradation. It has been hypothesized that p97 extracts proteins from membranes or macromolecular complexes to enable their proteasomal degradation; however, the complex nature of p97 substrates has made it difficult to directly observe the fundamental basis for this activity. To address this issue, we developed a soluble p97 substrate-Ub-GFP modified with K48-linked ubiquitin chains-for in vitro p97 activity assays. We demonstrate that WT p97 can unfold proteins and that this activity is dependent on the p97 adaptor NPLOC4-UFD1L, ATP hydrolysis, and substrate ubiquitination, with branched chains providing maximal stimulation. Furthermore, we show that a p97 mutant that causes inclusion body myopathy, Paget's disease of bone, and frontotemporal dementia in humans unfolds substrate faster, suggesting that excess activity may underlie pathogenesis. This work overcomes a significant barrier in the study of p97 and will allow the future dissection of p97 mechanism at a level of detail previously unattainable.
Keywords:
AAA ATPase; Cdc48; Npl4; Proteasome; Ufd1.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Vesicular Transport
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Adenosine Triphosphate / metabolism
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Frontotemporal Dementia / etiology
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Frontotemporal Dementia / genetics*
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Frontotemporal Dementia / metabolism*
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Humans
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Hydrolysis
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Intracellular Signaling Peptides and Proteins
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Kinetics
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Muscular Dystrophies, Limb-Girdle / etiology
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Muscular Dystrophies, Limb-Girdle / genetics*
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Muscular Dystrophies, Limb-Girdle / metabolism*
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Mutation
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Myositis, Inclusion Body / etiology
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Myositis, Inclusion Body / genetics*
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Myositis, Inclusion Body / metabolism*
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Nuclear Proteins / metabolism*
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Osteitis Deformans / etiology
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Osteitis Deformans / genetics*
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Osteitis Deformans / metabolism*
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Protein Unfolding
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Proteins / metabolism*
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Proteolysis
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Substrate Specificity
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Ubiquitin / metabolism
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Valosin Containing Protein / chemistry
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Valosin Containing Protein / genetics*
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Valosin Containing Protein / metabolism*
Substances
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Adaptor Proteins, Vesicular Transport
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Intracellular Signaling Peptides and Proteins
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NPLOC4 protein, human
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Nuclear Proteins
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Proteins
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Recombinant Fusion Proteins
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UFD1 protein, human
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Ubiquitin
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Adenosine Triphosphate
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VCP protein, human
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Valosin Containing Protein
Supplementary concepts
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Inclusion Body Myopathy With Early-Onset Paget Disease And Frontotemporal Dementia